JPS62138500A - Production of polypeptide - Google Patents

Abstract

PURPOSE:To efficiently obtain the titled compound of growth hormone-releasing factor short-chain peptide used for such as a remedy for microsomia, by reacting a condensate of specific peptide fragments having a protecting group with a specific peptide fragment having a protecting group successively, followed by deprotection. CONSTITUTION:A peptide fragment having a protecting group expressed by formula I is condensed with a peptide fragment having a protecting group expressed by formula II to obtain a condensate, which is then condensed with a peptide fragment having a protecting group expressed by formula III, further condensed with peptide fragments having a protecting group expressed by formulae IV and V successively and subjected to deprotection to obtain the aimed polypeptide expressed by formula VI. The condensation of these peptide fragments is preferably carried out by using a carbodiimide based condensing agent, additive, etc.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention provides a polypeptide (VI
).

H-Tyr-A la-Asp-A Ia-T Ie-
Phe-Thr-Asn-Ser-Tyr-Arg-L
ys-Val-Leu-Gly-Gln-Leu-Se
r -Asp -I Ie-Met -Ser -Ar
g -NH2(VI) This peptide is a growth hormone releasing factor (growth hormone releasing factor).
is a short-chain peptide derivative of GRF).

Like GRF, this peptide has a strong growth hormone secretion promoting effect, so it is not only effective for various growth hormone deficiency diseases such as dwarfism, but also can be used as an anti-ulcer agent, wound treatment agent, etc. It is extremely useful as a

Furthermore, this peptide has a growth hormone secretion promoting effect not only in humans but also in various animals, and is therefore useful for increasing the growth rate and degree of growth and milk yield in commercial animals.

[The problem that can be solved by the prior art and the invention] A solid-phase method is already known for producing the present peptide (Nature, 300°276, (19)
82) ).

Furthermore, the liquid phase method includes a stepwise method and a fragment condensation method. The fragment condensation method is an excellent method in terms of ease of product purification and peptide purity, but when performing fragment condensation, it is important to select a fragment from among many combinations of fragments. It becomes a challenge.

[Structure of the invention]

The present inventors conducted various studies on the solubility of peptide fragments in solvents, reactivity of peptide fragments, purification methods of products, etc. as a fragment condensation method.
They discovered that it is efficient to produce polypeptide (VI) by sequentially condensing the following five peptide fragments (I) to (V) starting from peptide fragment (I), and completed the present invention. .

Peptide fragment (I) Leu-Gln-Asp -I Ie -Met -S
er -Arg Nl+2 Peptide fragment (n) Gln-Leu-Ser-Ala-Arg-Lys -
Leu peptide fragment (I) Arg-Lys-Val-Leu-Gly peptide fragment (rV) I le -Phe -Thr-Asn-5er-Ty
r peptide fragment (V) Tyr -A la -Asp -A Ia (in the formula, all amino acids except glycine represent the L-configuration) That is, the present invention provides a peptide fragment (I
) and a peptide fragment (U) having a protecting group are condensed to obtain a condensate, and then this condensate is condensed with a peptide fragment (I) having a protecting group. The following is a method for producing a highly pure polypeptide (VI), which is characterized in that peptide fragments (IV) and (V) having protecting groups are sequentially condensed and then deprotected.

In the present invention, as a condensation method, a method that does not cause racemization is used, such as an azide method, a redox method, a diphenylphosphoryl azide method, a carbodiimide type condensation additive method, etc. Condensing agent +
It has been discovered that the additive method has the following advantages and is the most advantageous as an industrial manufacturing method.

(1) Operation is very simple. (Just add the raw materials and sample hollyhock to the solvent and stir.) (2) No need for cooling.

(3) There are almost no side reactions, and a highly pure target product can be obtained with simple post-treatment operations.

In the case of the carbodiimide type condensing agent/additive method, the condensation reaction of fragments is carried out, for example, as follows. That is, a peptide having a free carboxyl group and a reactive group protected by an appropriate protecting group and a peptide having a free amino group and a reactive group protected by an appropriate protecting group are dissolved in an appropriate solvent, and then subjected to carbodiimide-type condensation. Add the agent and additives and stir. At that time, if necessary, a suitable base may be added to keep the reaction mixture slightly alkaline.

Various solvents can be used, including dimethyl sulfoxide, dimethylformamide, hexamethylphosphoramide, dioxane, tetrahydrofuran, N-methylpyrrolidone, and mixtures thereof.

Examples of carbodiimide-type condensing agents include dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-
Dimethylaminopropyl)-carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, etc. can be used.

As additives in the carbodiimide type condensation additive method, for example, N-hydroxyconocarboxylic acid imide, 1-hydroxybenzotriazole, N-hydroxy-5-norbornene-2,3-dicarboximide, etc. can be used. . Further, the condensation reaction can normally proceed at a reaction temperature of about -20°C to 40°C without any problem.

As a means for removing the protecting group in this method, methods commonly used in the field of peptide synthesis, such as trifluoroacetic acid,
Methanesulfonic acid, p-)luenesulfone Ff12, phosphoric acid, hydrogen bromide-acetic acid, trifluoromethanesulfonic acid,
Examples include acid decomposition using hydrogen fluoride, hydrogenation using palladium black, palladium carbon, platinum, etc. as a catalyst, a reductive method using sodium in liquid ammonia, and a zinc-acetic acid method.

In acid decomposition it may be advantageous to add water, cation scavengers such as anisole, phenol, thiophenol, thioanisole, cresol, methyl ethyl sulfide.

Removal reactions of protecting groups by reduction or acid decomposition are usually carried out at -40℃~
There is no problem in proceeding at a reaction temperature of about 40°C.

Protecting groups used in the present invention include, for example, tosyl group, benzyloxycarbonyl group, tertiary butyloxycarbonyl group, trityl group,
p-methoxybenzyloxycarbonyl group, formyl group, trifluoroacetyl group, 0-nitrophenylsulfenyl group, 3-nitro-2-pyridinesulfenyl group, etc. Also, for protecting the carboxyl group of amino acids, for example, methyl ester, ethyl ester, isopropyl ester,
Lower alkyl esters and benzyl esters such as tertiary butyl ester, cyclopentyl ester, cyclohexyl ester, p-methoxybenyl ester, p-
Examples include protecting groups commonly used in the field of peptide synthesis, such as ditropendyl ester and phenacyl ester. In addition, protection of hydroxyl groups such as serine, threonine, and tyrosine is not necessarily essential, but if necessary, protection of hydroxyl groups such as acetyl group, benzyl group, 2,6-dichlorobenzyl group, benzyloxycarbonyl group, tertiary butyl group, etc. It can be protected by a hydroxyl protecting group. To protect the guanido group of arginine, for example, a nitro group, tosyl group, benzyloxycarbonyl group, adamantyloxycarbonyl group, isobornyloxycarbonyl group, mesitylene-2-sulfonyl group, etc. can be used.

Methionine can also be protected as a sulfoxide if desired.

The polypeptide (VI) produced by the present invention can be purified using methods commonly used in the field of peptide chemistry, such as ion exchange resin, partition chromatography, gel chromatography, and reversed-phase liquid chromatography, either singly or in combination. This can be done by using

The highly purified polypeptide (VI) produced as described above was tested in vivo using rats and in vitro using rat pituitary cells.
ro) showed a strong growth hormone releasing effect in the test.

In addition, peptide fragments (I) to (
The synthesis of V) can be carried out according to conventional peptide synthesis methods.

The known method includes, for example, M. Bodansky.
and Peptide Synthesis by M.A. Ondetti,
Interscience New York, 1
966; F, M, F inn and 'f, ,
l (Ofmann, The Proteins)
oteins), Volume 12, H, Neuroth,
Edited by R, L, Hil1, Δcademic Pre
ssInc, New York, 1976
Year: Nobuo Izumiya et al., Peptide Synthesis, round lid side, 1975; Complete Japanese Biochemistry, Biochemistry Experiment Course, Volume -, Chemistry of Proteins ■, Part ■, Yoshiaki Yajima, Peptide Synthesis, 19
Examples include the method described in 1977.

[Reference examples and working examples]

The present invention will be explained below with reference examples and examples.
This is just an example and does not limit the technical scope of the present invention in any way.

All of the amino acids used, except for glycine, are L-type, and amino acid abbreviations are represented by the first three letters of their names in accordance with general usage (with the exception of Asn-
asparagine, GIn=glutamine). Other abbreviations are as follows.

PMZ: p-methoxybenzyloxycarbonyl, Bo
c: t-butyloxycarbonyl, Mts: mesitylene-2-sulfonyl, ONp: p-nitrophenol ester, Bzl: benzyl, Cl2- Bzl: 2
, 6-dichlorobenzyl, Opac: 7 enacyl ester, 0Bzl: benzyl ester, Z: benzyloxycarbonyl, TFA: l-lifluoroacetic acid,
TosOH: p-toluenesulfonic acid, HOBt
: 1-Hydroxybenzotriazole, 5BCF: Nibutyl chloroformate, DCHA dicyclohexylamine, NMM: N-methylmorpholine, Et
iN: ) ethylamine, CHA dichlorohexylamine, DC'C dicyclohexylcarbodiimide, wsc: t-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, MeOH: methanol, TH
F: Tetrahydrofuran, EtOH: Ethyl alcohol, DMF Nidimethylformamide, DMS○ Nidimethylsulfoxide, Ac0NH4: Ammonium acetate.

HO5u: N-hydroxysuccinimide example I
Boc-Gln-Leu-Ser(Bzl)-Ala-
Production of Arg(Mts)-Lys(Zl-Leu-OH (compound 8) (1) Boc-Lys(Z)-Leu-Opac
Preparation of (Compound 1) 7 g of Boc-Lys (Z)-0821 is dissolved in 100 ml of ethyl acetate, and 5.76 g of NMM is added.

While cooling at −10° C., 7.81 g of 5BCF is added dropwise for 15 minutes. Then, after stirring for 15 minutes, TosOt(・H-Leu-
OPac 20. Dissolve Og in 50ml of DMF and
Add cooled to ℃. Next, 4.80 g of NMM was dissolved in 5 ml of DMF and added dropwise for 1 hour. After stirring at -10°C for 3 hours, the mixture was pressed in a dish for 3 hours while allowing natural humidity to rise. Add 300 ml of ethyl acetate, then wash with 5% citric acid solution, 5% sodium bicarbonate solution, and saturated saline in this order, dry over magnesium sulfate, and then evaporate the solvent. The residue is taken and washed with petroleum ether to obtain compound 1.

Yield 29.4 g (1,00%) Melting point 61-70°C [α:], -39.9° (C=1, MeOH) (
2) TosOHl(-Lys(Z)-Leu-OP
Preparation of ac (Compound 2) 27.0 g of Compound 1 is dissolved in 300 ml of acetonitrile. Add 16.8 g of p-toluenesulfonic acid hydrate and stir at room temperature for 3 hours. The precipitated crystals are collected by filtration.

The filtrate is evaporated and the residue is filtered and washed with petroleum ether. Both crystals collected by filtration are combined and dried to obtain Compound 2.

Yield 28.7g (94.9%) Melting point 139-143°C CtX], -16.3° (C=1, MeOH) (3
) PMZ-Arg(Mts)-Lys(Z)-Le
Preparation of u-OPac (Compound 3) PMZ-Arg(Mts) -0H-CHA 37.8
g to 500ml of ethyl acetate and 20ml of 5% citric acid in ice water.
01. After everything is dissolved, the layers are separated, the organic layer is dried over magnesium sulfate, and the solvent is distilled off. Dissolve the residue in THFlooml and add 5.69 g of NMM. While cooling at −10° C., 8.35 g of 5BCF is added dropwise for 15 minutes.

Then 32.1 g of compound 2 and N M M , 174
g was dissolved in a mixture of 20 ml of DMF and 30 ml of THF and added dropwise for 15 minutes under cooling. Stir for 3 hours while allowing the temperature to rise naturally. 500 ml of ethyl acetate and 200 ml of water are added and the layers are separated. The organic layer is washed sequentially with 5% citric acid water, 5% sodium bicarbonate water, and saturated saline. Dry blast over magnesium sulfate and evaporate the solvent. The residue is filtered and washed with petroleum ether to obtain compound 3.

Yield 46.1g (96.9%) Melting point 106-107°C [α), -24.3° (C=1, MeOH) (4)
Boa-Ala-Arg(Mts)-Lys(Z)
Preparation of -Leu-OPac (Compound 4) 19.7 g of Compound 3 is suspended in 20 ml of methylene chloride and 5 ml of anisole. Add 20 ml of TFA under ice cooling, stir for 1 hour, and then evaporate the solvent. After adding ether to the powder, it was filtered and dried to obtain a PMZ-free product. Next, this powder was dissolved in 50 ml of DMF and neutralized with 2.35 g of NMM.

On the other hand, 4.76 g of Boc-Ala-OH was added to 1 ml of ethyl acetate.
00ml and add 2.35g of NMM.

While cooling at −10° C., 3.19 g of 5BCF is added dropwise for 15 minutes. The PMZ-removed solution is added dropwise for 30 minutes under cooling. Stir for 3 hours while allowing the temperature to rise naturally.

Add 200 ml of ethyl acetate and 100 ml of water and separate the layers. The organic layer is washed sequentially with 5% citric acid water, 5% sodium bicarbonate water, and saturated saline. Dry smoke over magnesium sulfate and evaporate the solvent. The residue is filtered and washed with petroleum ether to obtain compound 4.

Yield 17.2g (86.9%) Melting point 100-104°C [α]. -39,4° (C=1.MeOH) (5)
Boa-3et(Bzl)-Ala-Arg(Mts)
Production of -Lys(Z)-Lau-OPac (Compound 5) 8.31 g of Compound 4 was dissolved in 25 ml of acetonitrile, 15.5 g of p-)luenesulfonic acid hydrate was added, and the mixture was stirred at room temperature for 2 hours. NMM8.22 under cooling to -10℃
Neutralize with g to obtain a de-Boc solution.

On the other hand, Boc -Set (Bzl) -0H2,88
Dissolve g in THFloml and add 99 g of NMMo. While cooling at -10°C, add 1.34 g of 5BCF and stir for 15 minutes. The above Boc-removed solution was added dropwise for 15 minutes, and the mixture was stirred for 3 hours while allowing the temperature to rise naturally.

Add 300 ml of ethyl acetate and 100 ml of water and separate the layers. The organic layer was mixed with 5% citric acid water and 5% hydrogen carbonate l-U.
Rinse with rum water and then water. After evaporating the solvent, the residue is collected by filtration and washed with ether to obtain Compound 5.

Yield 10.5g (89.7%) Melting point 120-123℃ CCx]n 2R,7 (C=1, MeOH) (
6) Boc-Leu-Ser(Bzl>-Ala
-Arg(Mts)-Lys(Z)-Leu-OPac
Preparation of (Compound 6) 7.5 g of Compound 5 was suspended in 50 ml of acetonitrile, 11.8 g of p-)luenesulfonic acid hydrate was added, and the mixture was stirred at room temperature for 1 hour. NMM6.32 under cooling to -10℃
Neutralize with g to obtain a de-Boc solution. On the other hand, Boc-L
87 g of eu -OH.H201 was dissolved in 5 ml of ethyl acetate and dried over magnesium sulfate. NMM O, 7
Add 5g of 5BCF, and while cooling to -10°C, add 1.03g of 5BCF and stir for 15 minutes. The previous de-Boc compound solution was cooled 1
Add dropwise for 5 minutes and stir for 3 hours while allowing the temperature to rise naturally. Add 300 ml of ethyl acetate and 100+n+ of water and separate the layers. The organic layer was dissolved in 5% citric acid water and 5% hydrogen carbonate) I
Wash with water and water in that order.

The solvent was distilled off, the residue was collected by filtration, and washed with ether to obtain compound 6.
get.

Yield 6.98g (85.0%) Melting point 164-165℃ [(2) o-27.8° (C=1.MeOH) (7)
Boc-Gln-Leu-Ser(Bzl)-A
la -Arg(Mts)-Lys(Z)-Leu-O
Preparation of Pac (compound?) 5.00 g of Compound 6 was added to 50 ml of acetonitrile, 7.24 g of p-toluenesulfonic acid hydrate was added, and the mixture was stirred for 1 hour. After neutralizing by adding 3.84 g of NMM, acetonitrile is distilled off. D MF 10m of the residue
1 and add 2.80 g of Boc-Gln-ONp. Add NMM and stir at room temperature for 16 hours while keeping the mixture slightly alkaline (pH 7-8). The reaction mixture is added dropwise to 200 ml of water. The deposited precipitate is collected by filtration to obtain crude crystals.

The crude crystals were clouded with 200ml of acetic acid and heated to 50°C for 30 minutes.
Stir for a minute. Obtain Compound 7 by removing the filter.

Yield 4.57g (83.2%) Melting point 204-208℃ [α], -20.1° (C=1.DMSO) (8)
Boa-Gln-Leu-5er (Bzl)-Ala
-Arg(Mts) -Lys(Z) -Leu-O
Preparation of H (Compound 8) 3.50 g of Compound 7 is dissolved in 50 ml of acetic acid.

Add 3.0 g of zinc powder and stir at room temperature for 2 hours.

After filtering off insoluble matter, the solvent is distilled off. The residue is washed with water, filtered and smoked to obtain Compound 8.

Yield 3.07g (95.5%) Melting point 192-194°C [α], -14.3° (C=1.DMSO) Reference Example 2
PMZ-Arg(Mts)-Lys(Z)-Val-
Production of Leu-Gly-OH (Compound 13) (1) Production of Boc-Leu-Gay-OPac (Compound 9) 16.7 g of Boc-Leu-OH-HzO was added with 400 ml of ethyl acetate and 200 ml of saturated brine and branched. .

The upper layer was washed with 200 ml of saturated saline, and after drying with magnesium sulfate, the solvent was distilled off, and the resulting residue was diluted with DMF1.
Dissolve in ooml TosOH・H-Gly-OPac
23.3g, HOBt 9. Add Og, cool to -15°C, and dropwise add 4 g of WSClo. After 3 hours, the mixture was returned to room temperature and stirred for 24 hours.

Add 500 ml of toluene and 300 ml of water to the reaction solution and separate the liquid. The upper layer is water, 5% hydrogen carbonate water, 5
% citric acid solution and saturated saline solution, and dried over magnesium sulfate, the solvent was distilled off, and 100 ml of ether was added to the resulting residue, followed by crystallization by adding 400 ml of hexane while stirring. Collect the crystals, wash them with hexane, and dry-blast them to obtain compound 9.
get.

Yield 24.2g (93.5%) Melting point 108-110℃ Ccr], -29°5° (C=1, MeOH) (
2) Boc -Val-4eu -G Iy -O
Preparation of Pac (Compound 10) 18.9 g of Compound 9 was dissolved in 190 ml of acetonitrile, 4 g of TosOH.H-035 was added, and after stirring at room temperature for 1.5 hours, the solvent was distilled off, and the resulting residue was dissolved in THF90.
ml and neutralized with 6 ml of EtJ19 while cooling with water.

In this solution, 6.9 g of HOBt', Boc-Val-O
Add 11.1 g of H and dropwise add 8.0 g of W SC. 2
After that time, the mixture was returned to room temperature and stirred for 24 hours. Add 500 ml of ethyl acetate and 300 ml of water to the reaction solution and separate the layers. The upper layer is water, 5% sodium bicarbonate water, 5% citric acid water,
The mixture is washed with saturated brine, the solvent is distilled off, and 500 ml of petroleum ether is added to the resulting residue for crystallization. The crystals are collected by filtration, washed with petroleum ether, and dried to obtain Compound 10.

Yield 22.0g <93.6%) Melting point 146-147°C CCr], -54,8° (C=1, MeOH) (3
) Boc-Lys(Z)-Val-Leu-Gl
Production of y-0Pac (Compound 11) 21.5 g of Compound 10 was dissolved in 66.5 ml of TFA, stirred at room temperature for 40 minutes, and then dissolved in 140 ml of THF.
1 and neutralized with 113.3 ml of E ta N under water cooling. Add HOBt 6JgSBoc-Lys (Z
)-OH 17.8g and DMF 50ml were added and WSe2.
Add 3g dropwise. After 2 hours, the mixture was returned to room temperature and stirred for 24 hours. Add 1.200ml of ethyl acetate and 500ml of water to the reaction solution.
Add and separate the liquids. The upper layer was washed with water, 5% aqueous sodium bicarbonate, 5% aqueous citric acid, and saturated saline in this order, the solvent was distilled off, and the resulting residue was mixed with 2 ooml of ether and x -
Add 400 ml of TeJl and crystallize. The crystals are scaled, washed with petroleum ether, and dried to obtain crude crystals. The crude crystals were suspended in 200 ml of acetone, stirred at 40°C for 30 minutes, added with 400 ml of ether, returned to room temperature, collected by filtration, and dried to obtain Compound 11.

Yield 27.2g (80.1%) Melting point 189-191°C [α), -21.7° (C=1., DMF) (4,)
PMZ-Arg(Mts)-Lys(Z)-Val
-Leu -G ly -OPac (Compound 12) 115.5 g of the compound was dissolved in 11.3 ml of TFA, stirred at room temperature for 40 minutes, and then dissolved in 24 ml of T H-F.
1 and neutralized with 20.2 ml of E b N under ice cooling.

To this solution, 1.1 g of HOBt, PMZ-Arg (Mt
s) Add 4.5 g of -OH and 24 ml of DMF WSC
Drop 2 g of the solution. After 2 hours, the mixture was returned to room temperature and stirred for 244 hours. The reaction solution was concentrated and added dropwise to 240 ml of water at 5°C or below to crystallize. Filter the crystals, wash with water and ether, and dry to obtain coarse crystals. Coarse crystals in MeOH40
ml, stirred at 40°C for 30 minutes, filtered, and dried to obtain Compound 12.

Yield 7.1g (84.8%) Melting point 200-203℃ [α), -10.7° (C=1.DMF) (5) P
MZ-Arg(Mts)-Lys(Z)-Val-Le
Production of u-Gly-OH (compound 13) Compound 12 4
．． Dissolve 9g in A co 8100ml and add zinc powder 7,
Add 5 g in small portions at 10 minute intervals and stir for 3 hours. After filtering through Celite and distilling off the solvent, add 1 ether to the resulting residue.
Add 00ml and crystallize. The crystals are collected by filtration, washed with ether, and dried to obtain Compound 13.

Yield 4.4g (99.8%) Melting point 195-201℃ [α) o-11,1° (C-1, DMF) saturation example 3
Boc-Ile-Phe-Thr-Asn-Ser-T
Production of yr-OH (compound 15) (1) Boc-Ile-Phe-Thr (Bzl
) -Asn-3et (Bzl) -Tyr (B
zl) -0Bzl (Compound 14) Production Boc-Phe-Thr(Bzl) -Asn-3et
(Bzl) -Tyr (Bzl) -0Bzl [Melting point 197-199°C, [α]? +i, 3° (C=1.
44.7 g of DMF) was suspended in 270 ml of acetonitrile, and a solution of 39.4 g of methanesulfonic acid dissolved in 138 ml of acetonitrile was added dropwise at 5°C, and the suspension was suspended at room temperature for 1
Stir for an hour. Under water cooling, DMF480ml, EtaN
After dropping 37.3g, HOBt6.70g, Bo
82 g of c-T Ie-OH.1/2H2010 is added, followed by dropwise addition of 2.7 g of WSe, and the mixture is stirred for 30 minutes under water cooling and then for 2.5 hours at room temperature.

At the end of the reaction, the reaction solution was poured into 4.8 ml of water under water cooling, and the resulting precipitate was collected by filtration, washed with water, and then dried under reduced pressure to obtain crude compound 1 50.8 ml.
I got g. Then, 150.8 g of the crude compound was dissolved in 740 mL of acetonitrile, stirred at room temperature for 1 hour, collected by filtration, washed with acetonitrile, and then dried under pressure to obtain 45.4 g of Compound 14.

Yield 45.4g (92%) Melting point 210-211℃ [α]′L knee 2.0° (C=1.1.DMF) (2)
Boc -Ding 1e -\'he-Thr -
Production of Asn-Ser -Tyr-OH (compound 15) 40.1 g of compound 1,4 was DMF 1.200 m
1. Dissolved in 600ml of acetic acid, 5% Pd-C40.4g
Catalytic reduction is carried out at 40° C. for 5 hours at normal pressure in the presence of. The catalyst was removed by filtration through Celite, and the filtrate was concentrated under reduced pressure to obtain an oil. Ether was added to this oil to crystallize it, which was collected by filtration, washed with ether, and dried to obtain Compound 15.

Yield 22.7g (80.7%) Melting point 199℃ (decomposition) [α]τ+1.9° (C=1.1.DMF) Reference example 4
Boc-Tyr (C12-Bzl)-81a-A
Production of sp(OBzl)-Ala-OH (compound 19) (1) Boc-Asp(OBzl)-Ala-O
Preparation of Pac (compound 16) TosOH-H-Ala-OPac 100g, Bo
c-Asp(OBzl) OH93.7g and HOBt
Dissolve 39.2 g in DMF 750 mA. WSC4
5.0 g was added dropwise at -10°C for 1.5 hours and stirred at the same temperature for an additional hour.

Ethyl acetate 1β and 600 ml of saturated saline were added and the layers were separated. The organic layer was dissolved in 5% citric acid water and 8% hydrogen carbonate)
Wash with IJum water and water in that order. Dry smoke over magnesium sulfate and evaporate the solvent. The residue is collected by filtration and washed with hexane to obtain Compound 16.

Yield 114g <84.4%) Melting point 107~
109°C (α)o 36,7° (C=1, MeOH) (2
) Boc-Ala-Asp(OBzl)-Al
Production of a-0Pac (Compound 17) 40.0 g of Compound 16 was dissolved in 240 ml of acetonitrile.
59.3 g of P-toluenesulfonic acid hydrate was added to the solution, and the mixture was pressed for 1 hour at room temperature. EtaN under cooling to -10℃
Neutralize with 31.5 g to obtain a de-Boc solution.

On the other hand, 16.2 g of Boc-A Ia-OH was dissolved in 80 ml of ethyl acetate, and 66 g of NMML and 11.8 g of 5BCF were added dropwise over 5 minutes while cooling at -10°C. After stirring for another 15 minutes at the same temperature, the BOC-removed solution was added dropwise for 15 minutes and the mixture was stirred for 3 hours while allowing the temperature to rise naturally. Add 600 ml of ethyl acetate and 300 ml of saturated brine and separate the layers. The organic layer is washed sequentially with 5% citric acid water, 8% sodium bicarbonate water, and water. After drying over magnesium sulfate, the solvent was distilled off under reduced pressure. The residue was recrystallized from ethyl acetate-hexane to obtain Compound 17. Yield 42.6
g (93,6%) Melting point 145-147°C [α), -51,0° (C=1.DlvlF) (3)
Boc-Tyr (CI2- Bzl) -A
Preparation of Ia-Asp(OBzl)-Ala-OPac (Compound 18) Compound 1? 20.0 g of acetonitrile Zoom
1. P-) Luenesulfonic acid hydrate 26
．． Add 0g and stir at room temperature for 1 hour. The mixture was cooled to −10° C. and neutralized with 13.8 g of Et3N to obtain a Boc-free solution. On the other hand, Boa -Tyr (CI2- Bzl) -O
Dissolve H16.6g in THF50g and NMM3.80g
Add. While cooling to -10°C, add 5.16 g of 5BCF and press the plate for 15 minutes. The above Boc-removed solution was added dropwise for 15 minutes, and the mixture was stirred for 3 hours while allowing the temperature to rise naturally. ethyl acetate 5
00 ml and 200 ml of saturated saline were added and the layers were separated. The organic layer is washed sequentially with 8% sodium bicarbonate and water. Dry over magnesium sulfate. Hexane 50
Add 0ml. Compound 18 is obtained by filtering the precipitate and washing with hexane.

Yield: -25.0g Melting point: 174-176°C [α] -20.7° (C=1.DMF) (4) B
oc-Tyr (CI2- Bzl) -A Ia-A
Preparation of sp(013zl) -Ala-○H (Compound 19) 20.0 g of Compound 18 is dissolved in 240 ml of acetic acid.

Add 219 g of zinc powder and stir at room temperature for 1 hour. After removing the insoluble matter, the solvent is distilled off. The residue is washed with water, filtered and dried. Compound 19 is obtained by recrystallization from ethyl acetate-hexane.

Yield 14.2g (81.3%) Melting point 132-138°C [α]π-14,2° (C=1.DMF) Example I H-Tyr -A la -Asp -A la-I
Ie -Phe -Thr-Asn-5er-Tyr-
Arg-Lys-Val-Leu-G Iy-G
In -Leu -Ser -A la -Arg -
Lys -Leu -Leu-G In -Asp -
I le -Met -Ser -Arg -N I2
Production of (polypeptide ■) (1) H-Leu-G
ln-Asp(OBzl)-Ile-Met(0)-
Preparation of 3et(Bzl)-Arg(Mts)-NI2 (Compound 20) Boc-Leu-Gln-Asp(OBzl)-I
le-Met(○) -Ser (Bzl)-8rg(
Mts)-NI2 (melting point 220-222°C [α].-1
0.7° (C:1, DMSO): 15.0g of TFA3
Dissolve in 0 ml and stir at room temperature for 1 hour. 500ml water
54.2 ml of E t=N was previously dissolved in the solution, cooled on ice, and the TFA solution was added dropwise. Collect the precipitate by filtration,
Compound 20 is obtained by washing with water and drying.

Yield 4.55g (98.3%) Melting point 203-210℃ [α] -7.0° (C=1.DMSO) (2) B
oc-Gln-Leu-8,er(Bzl)-Ala
-Arg(Mts)-Lys(Z)-Leu-Leu-
Gln-Asp(OBzl)-Ile-Met(0)-
Ser (Bzl) -Arg (Mts) -N I
2 (Compound 21) Production Compound 8 2,60. g, compound 202.54 g and H
OS u 272mg DMF5ml DMSO2,5
ml of mixed solvent. Add 36 mg of DCCA. Add NMM and stir at room temperature for 24 hours while keeping the mixture slightly alkaline (pH 7-8). The reaction mixture is added dropwise to 50 ml of water. The deposited precipitate was collected by filtration, suspended in 30 ml of MeOH, and stirred at 50°C for 30 minutes. Compound 21 is obtained by filtering and drying.

Yield 4.16g (816%) Melting point 270-271℃ [α]τ-17.2° (C=1.DMSO) (3)
H-Gln-Leu-Ser(Bzl)-Ala
-Arg(Mts) -Lys(Z) -Leu-Le
u -Gln-Asp(OBzl) -I le -M
et (0)-Ser(Bzl)-Arg(Mts)
-Production of NI2 (Compound 22) 3.50 g of Compound 2j was dissolved in 35 ml of TFA and stirred for 30 minutes under water cooling. Add EtaN66 to 500ml of water in advance.
The +n I solution is cooled on ice, and the TFA solution is added dropwise. The precipitate was collected, washed with water, and dried to obtain compound 22.
get.

Yield 3.31. g (98,2%) Melting point 264-267℃ [α]? 219° (C=1, DMSO) (4)
PMZ-Arg(Mts)-Lys(Z)-Val-
Leu -Gly -Gln -Leu -Ser (
Bzl) -Δla-Arg(Mts)-Lys(Z)
-Leu-Leu-Gln-Asp(OBzl) -I
le-Met(0)-Ser(Bzl)-Arg(Mt
s) Preparation of -NI2 (Compound 23) 1.87 g of Compound 13 and 325 mg of HOB t are dissolved in a mixed solvent of 20 ml of DMSO and DMFloml. Add 372 mg of D CC and 3.0 g of Compound 22. Stir at room temperature for 10 hours while keeping the mixture slightly alkaline (pH 7-8) with NMM.

The reaction mixture was added dropwise to 30 ml of water, and the precipitate was collected by filtration. MeOH30m1! I! 11. Stir at 50°C for 30 minutes. Compound 23 is obtained by filtering and drying.

Yield 3.87g (91.5%) Melting point 275-278℃ [α]2D 19.9° (C=1.DM, 5O) (5
) H-Arg(Mts)-Lys(Z)-Va
t -Lau-Gly-Gln-Leu-Ser(Bz
l) -ΔIa-Arg(Mts)-Lys(Z)-L
eu-Leu-Gln-Asp(OBzl)-Ile
-Met(○) -Ser(Bzl) -Arg(Mt
s) Production of -NH2 (Compound 24) 3.50 g of Compound 23 was dissolved in 15 ml of TFA and 5 ml of anisole and stirred for 30 minutes under water cooling. water 300m1
Dissolve 4 ml of EtsN2B in advance, cool on ice, and add the TFA solution dropwise. The deposited precipitate was collected by filtration, washed with MeOH, filtered, and dried to obtain Compound 24.

Yield 3.03g (89.1%) Melting point 280-283℃ [α)? -2s, o° (C=1.DMSO) (+3)
Boc-Ile-Phe-Thr-As
n-Ser-Tyr-Arg(Mts)-Lys
(Z)-Val-1, eu-Gly-Gln-Leu
-Ser (Bzl) -Ala -Arg (Mt
s) -Lys (Z) -Leu -Leu-Gl
n-Asp (OBzl) -I Ie -Met(0
) -Set (Bzl) -Arg (Mts) -
Preparation of NH, (Compound 25) Compound 24 735 mg Compound 15 281 mg.

92 mg of HOBt-monohydrate was dissolved in 7.5 ml of DMSO, N
-Dissolved in 7.5 ml of methylpyrrolidone and subjected to WSC under water cooling.
70 mg of hydrochloride was added and stirred for 244 hours under water cooling. After the reaction was completed, 150 ml of water was added to the reaction solution, and after stirring for 30 minutes, the deposited precipitate was collected by filtration, washed with water, and dried under reduced pressure to obtain compound 2.
Got 5.

Yield 769mg (83.8%) Melting point 128-140℃ [α], -11.5° (C=1.L DMSO')
(7) Boc-Tyr(c12-Bzl)-A
la-Asp(OBzl) -A Ia -I Ie
-Phe -Thr-Δsn-Ser -Tyr -
Arg, (Mts) -Lys (Z) -Val-
Leu -G Iy-G In -Leu-Ser (
Bzl)-Ala-Arg(Mts)-Lys(Z)-
Leu-Leu-Gln-Asp(OBzl)-Ile
-Met(0)-3et(Bzl)-Arg(Mts)
-Production of NH2 (Compound 26) 910 mg of Compound 25 was cooled with TFA 3.39
Add m1 and stir for 1 hour under water cooling. Subsequently, 3.9 ml of DMF, 7.36 ml of Et, and N were added dropwise to the mixture under ice-cooling with sodium chloride, followed by addition of 24 ml of water, and the mixture was stirred for 15 minutes.

The deposited precipitate was collected by filtration, washed with water, and dried under reduced pressure to obtain 910 mg of Compound 25 without Boc.

910m of de-Boc compound of compound 25 obtained by the above treatment
g, Compound 19 173mg1HOBt・-hydrate 67
4.mg of DMSO, 5ml of N-methylpyrrolidone.
The mixture was dissolved in 5 ml, and 46 mg of WSC/hydrochloride was added under ice-cooling, followed by stirring for 30 minutes under water-cooling and then at room temperature for 2 hours. After the reaction was completed, 90 ml of water was added to the reaction solution, and the precipitate precipitated was collected by filtration, washed with water, and then dried under reduced pressure to obtain Compound 26.

Yield: 936mg (88.5%) Melting point: 130
~145℃ [α]. −10,8°(, C=1.0.DMSO)(8
) Production of polypeptide (VI) Compound 26 300+y+g and m-cresol 287μ
β, thioanisole 323μ! 302 μA of trichloromethylsilane, 1.8 ml of TFA, and 304 μβ of trifluoromethanesulfonic acid were added, and the mixture was stirred for 2.5 hours under ice cooling. The reaction solution was added to 50 ml of ether, and the precipitate was placed and washed with ether. The obtained precipitate was dissolved in 90 ml of 5% acetic acid and 9 ml of 1.2-mercaptoethanol.
The mixture was dissolved in 1 ml of hydroxide, adjusted to pH 9 with 1-U hydroxide rum water, stirred for 1 hour, and adjusted to pH 5 with acetic acid. This solution was subjected to reverse phase column chromatography (YMC-GEL 0DSS-15/3
0) Adsorbed on 20 x 300 fence, water, 0, I
After washing in the order of M Ac0NH471<(pH5,0), 35% acetonitrile-65%0.1! ,I Ac
Eluted with NH and water (pH 5, 0). 600m1～
A 650ml elution fraction was collected and concentrated. This concentrated solution was again subjected to reverse phase column chroma) (YMC-GE LOD S
S-15/30) Adsorbed on 20x300mm, washed with 1% acetic acid water, then 22% acetonitrile-78%
Elution was performed with 1% acetic acid water. Elution fractions of 700 ml to 800 ml were collected, concentrated under reduced pressure, and freeze-dried to obtain 56 mg of the target polypeptide (VI).

[:α], -65° (C=0.1.H2O) Example 1
Compound 2526 and polypeptide (VI
) Amino acid analysis values are shown in Table 1. Analysis values are based on 6N hydrochloric acid hydrolysis.

・Theoretical values are in parentheses

Claims (2)

[Claims] (1) A peptide fragment (I) having a protecting group is condensed with a peptide fragment (II) having a protecting group to obtain a condensate, and then this condensate is condensed with a peptide fragment (III) having a protecting group. A method for producing a polypeptide (VI) having the following amino acid sequence, which comprises sequentially condensing peptide fragments (IV) and (V) having protecting groups and then deprotecting them. [There is an amino acid sequence] (VI) However, the peptide fragments (I) to (V) are represented by the following formula. Peptide fragment (I) Leu-Gln-Asp-Ile-Met-Ser-A
rg-NH_2 Peptide fragment (II) Gln-Leu-Ser-Ala-Arg-Lys-L
eu Peptide fragment (III) Arg-Lys-Val-Leu-Gly Peptide fragment (IV) Ile-Phe-Thr-Asn-Ser-Tyr peptide fragment (V) Tyr-Ala-Asp-Ala (all except glycine in the formula The amino acids in represent the L-form.) (2) Peptide fragment (II) with a protecting group, (
The method for producing polypeptide (VI) according to claim 1, which uses a carbodiimide condensing agent + additive method as the condensation method for III), (IV) and (V).